Synthetic analogs of bacterial quorum sensors

ABSTRACT

Bacterial quorum-sensing molecule analogs having the following structures: 
                         
and methods of reducing bacterial pathogenicity, comprising providing a biological system comprising pathogenic bacteria which produce natural quorum-sensing molecule; providing a synthetic bacterial quorum-sensing molecule having the above structures and introducing the synthetic quorum-sensing molecule into the biological system comprising pathogenic bacteria. Further is provided a method of targeted delivery of an antibiotic, comprising providing a synthetic quorum-sensing molecule; chemically linking the synthetic quorum-sensing molecule to an antibiotic to produce a quorum-sensing molecule-antibiotic conjugate; and introducing the conjugate into a biological system comprising pathogenic bacteria susceptible to the antibiotic.

RELATED APPLICATIONS

This application is a divisional of copending U.S. patent applicationSer. No. 12/551,994 entitled “Synthetic Analogs of Bacterial QuorumSensors,” filed Sep. 1, 2009, now allowed, which is incorporated byreference herein.

STATEMENT OF FEDERAL RIGHTS

The United States government has rights in this invention pursuant toContract No. DE-AC52-06NA25396 between the United States Department ofEnergy and Los Alamos National Security, LLC for the operation of LosAlamos National Laboratory.

FIELD OF THE INVENTION

The present invention relates to synthetic analogs of bacterial quorumsensing molecules, and methods of use thereof.

BACKGROUND OF THE INVENTION

Interbacterial signaling, commonly referred to as quorum sensing (QS)enables bacteria to coordinate their behavior in order to function as agroup. Using diffusible chemical signals to initiate a concertedpopulation response depends on the population reaching a thresholdnumber or “quorum”. Most microbes require a “quorate population” tomanifest an infection in its target host. This bacterial intercellularcommunication system relies on the production and release of QSmolecules that control the expression of multiple target genes whichplay is pivotal role in virulence and pathogenicity in the host. Themost intensively investigated QS signal molecules are acyl-homoserinelactones (AHLs) synthesized by many quorum-sensing, secreting bacteria.AHLs are used to regulate infection, virulence and survival functions.Different bacterial species can produce different AHLs. The basicstructure of AHLs consists of a homoserine lactone ring adjoined with anN-acyl chain, ranging in length from 4 or 14 carbons, which may besaturated or unsaturated and may or may not contain a hydroxy- oroxo-group at the 3-carbon position.

To date, AHL-dependent QS circuits have been identified in a wide rangeof gram-negative bacteria, one example of which is Pseudomonasaeruginosa, where the QS circuits regulate various functions to survivein the host. The severity and diversity of infections caused by P.aeruginosa, is in part due to its ability to produce a plethora ofenvironment-dependent virulence factors but also due to itsrecalcitrance to antibiotic treatment when growing in biofilm.

The accepted clinical intervention strategy in bacterial infections istreatment with antibiotics. However, currently prescribed small moleculeantibiotics have limitations, especially in advanced infectious stateswhen systemic application cannot provide the required local dose ofantibiotics necessary for effective bactericidal action due tocompromised half-life of the drug. The efficacy of the antibioticdepends on factors such as selective toxicity, bioavailability of thedrug and penetration into the target bacteria. Some very effectiveantibacterial compounds are unacceptable for human use as they are toxicat their prescribed doses, chiefly due to the fact that current deliveryregimens are systemic, thus requiring a whole body dosage to achievenecessary local concentrations. There exists an ongoing need, therefore,for effective treatment of bacterial infections without thedisadvantages resulting from traditional systemic application ofantibiotics.

SUMMARY OF THE INVENTION

The present invention meets the aforementioned need by providingsynthetic analogs of bacterial quorum sensing molecules, specifically,by providing analogs of an autoinducer of Pseudomonas quorum sensingsystem, butanol homoserine lactone (AHL-2). It is believed that theseanalogs may meet the aforementioned need by either perturbation ofcritical survival mechanisms by interference with QS-signaling and/or byprecise delivery of an antibiotic to its target. In other words, theanalogs may act as agonists or antagonists, in that AHL-antagonists mayperturb QS-based bacterial communication and consequently attenuatebacterial virulence, while AHL-agonists may be used to manipulateexisting bacterial mechanisms for the targeted delivery and enhanceduptake of antibiotics conjugated to analogs of self-derived moleculessuch as AHLs.

In addition, it is believed that AHL-agonists could be used to inducethe premature expression of immunogenic molecules, therefore exposingthe bacteria to the host immune system at an early state of infection.The basis for this belief is the observation that P. aeruginosa evadesdetection by the immune system by rapidly down-regulating thetranscription and expression of flagellin, a highly immunogenicmolecule.

The following describe some non-limiting embodiments of the presentinvention.

According to a first embodiment of the present invention, a method ofreducing bacterial pathogenicity is provided, comprising providing abiological system comprising pathogenic bacteria which produce a naturalquorum-sensing molecule; providing a synthetic bacterial quorum-sensingmolecule having the structure:

and introducing the synthetic quorum-sensing molecule into thebiological system comprising pathogenic bacteria.

According to another embodiment of the present invention, a method ofreducing bacterial pathogenicity is provided, comprising providing abiological system comprising pathogenic bacteria which produce a naturalquorum-sensing molecule; providing a synthetic bacterial quorum-sensingmolecule having the structure:

and introducing the synthetic quorum-sensing molecule into thebiological system comprising pathogenic bacteria.

According to yet another embodiment of the present invention, abacterial quorum sensing molecule analog is provided which has thestructure:

According to yet another embodiment of the present invention, abacterial quorum-sensing molecule analog is provided which has thestructure:

According to yet another embodiment of the present invention, a methodof targeted delivery of an antibiotic is provided, comprising providinga synthetic quorum-sensing molecule; chemically linking the syntheticquorum-sensing molecule to an antibiotic to produce a quorum-sensingmolecule-antibiotic conjugate; and introducing the conjugate into abiological system comprising pathogenic bacteria susceptible to theantibiotic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures and synthesis of (S)—N-butyryl homoserinelactone (AHL-2) (Scheme 1), (S)—N-butyryl homocysteine thiolactone(QS0108) (Scheme 2), and (S)—N-Thiobutyryl homocysteine thiolactone(QS1207).

FIG. 2 shows the synthesis of one example of a QS analog-antibioticconjugate

FIG. 3 shows the quantification by CFU (colony forming units) count ofpersister bacterial cells from biofilms grown on collagen coatedcoverslips (A), free Cip coated (B) or QS0108-Cip conjugate coated (C)coverslips for 48 hrs. Y-axis represents CFU log/ml.

FIG. 4 shows a synthesis of a selenium-containing quorum-sensing analog,N-(2-oxo-tetrahydro-furan-3-yl)-selenobutyramide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel, synthetic bacterial quorum-sensinganalogs, and methods of using these analogs to reduce bacterialpathogenicity and for targeted delivery of antibiotics. Surface adheredbacterial colonies or biofilms are an important problem in medical andfood industries. Bacteria use a chemical language to monitor theirquorum and to express virulence factors, which eventually help them incolonization and manifestion of an infection. For example, the LasR-LasIand Rh1R-Rh1I quorum sensing systems of Pseudomonas aeruginosa controlexpression of virulence factors in a population density-dependentfashion. The synthetic analogs described herein have been shown to serveas AHL-agonists by promoting bacterial growth, virulence factorproduction and biofilm formation. Without wishing to be limited bytheory, it is believed that these responses are due either to rapid‘threshold concentrations’ reached by the addition of extraneous AHLanalogs or to the preferential and enhanced uptake of these molecules bythe bacteria. It is further believed that coupling of an antibiotic tothese analogs may facilitate targeted delivery of the antibiotic throughan otherwise impenetrable biofilm barrier.

Herein, “pathogenic bacteria” means a bacteria capable of causingdisease in a host organism when present in sufficient numbers (i.e., a“quorum”).

Herein, “synthetic” means a molecule not produced naturally by anorganism or found in nature, as opposed to a “natural” molecule, whichas used herein means a molecule produced by an organism or biologicalsystem.

Herein, “analog” means a synthetic molecule capable of performing one ormore of the biological functions that a natural counterpart wouldperform, such as controlling the expression of target bacterial genes,virulence factor production and biofilm formation. The analog may differfrom the natural molecule by a single atom or by multiple atoms.

The synthetic bacterial quorum-sensing molecule of the presentinvention, also termed “synthetic analogs,” “quorum-sensing analogs,” or“QS-analogs,” are analogs of a natural acyl-homoserine lactone depictedin (I), and biologically mimic the behavior thereof.

The synthetic analogs of the present invention comprise a lactone ring,either unsubstituted or substituted with a sulfur atom and a side chaineither unsubstituted or substituted with a sulfur atom or a seleniumatom. Non-limiting examples of suitable bacterial quorum-sensingmolecules of the present invention include those depicted in (II), (III)and (IV):

One aspect of the present invention is providing a method of reducingbacterial pathogenicity. As used herein, “reducing bacterialpathogenicity” means that the number of bacteria in a biological systemafter exposure to a synthetic analog is reduced as compared to a similar(control) biological system which is not exposed to a synthetic analog,or alternatively that the number of bacteria in a biological, systemexposed to the synthetic analog increases as compared to a controlbiological system which is not exposed to a synthetic analog, but doesnot result in the appearance of measurable indicators of pathogenicity,such as biofilm formation. The method of reducing bacterialpathogenicity comprises providing a biological system which comprises atleast one type of pathogenic bacteria which produce a naturalquorum-sensing molecule. The biological system may include culturedbacteria, a biological sample, or an organism, including an animal orhuman. The pathogenic bacteria may be any bacteria which produce anatural quorum-sensing molecule as defined herein. In one embodiment,the pathogenic bacteria are Pseudomonas aeruginosa (P. aeruginosa),Yersinia pestis, Yersinia enterocolitca, Yersinia pseudotuberculosis,Burkhotderia cepecia, and combinations thereof. In another embodiment,the pathogenic bacteria are Pseudomonas aeruginosa.

The method further comprises the step of providing a synthetic bacterialquorum-sensing molecule, as described herein. The synthetic bacterialquorum-sensing molecule may be introduced into the biological system bya variety of means that would be known to one of skill in the art, suchas by placing the synthetic molecule into a suitable carrier andadministering topically, orally, via inhalation or via injection.

Another aspect of the present invention is to provide a method oftargeted delivery of an antibiotic to a cell or area of interest in abiological system. By “targeted delivery” is meant delivery of theantibiotic to, for example, a pathogenic bacteria or area of infection,as opposed to systemic delivery of the antibiotic to the entireorganism. The method comprises the step of providing any one of thesynthetic bacterial quorum-sensing molecules, as described herein anddepicted in (II), (III), or (IV). The synthetic quorum-sensing moleculeis then chemically linked, whether covalently or non-covalently, to asuitable antibiotic, resulting in a conjugate comprising thequorum-sensing molecule and the antibiotic (“quorum-sensingmolecule-antibiotic conjugate,” or “conjugate”). The conjugate is thenintroduced into a biological system which comprises one or more types ofpathogenic bacteria susceptible to the antibiotic, wherein “susceptible”means that the antibiotic is known to reduce the number of live bacteriain systems wherein the antibiotic is not linked to a quorum-sensingmolecule. It is understood that the pathogenic bacteria naturallyproduce a quorum-sensing molecule substantially similar to the naturalquorum-sensing molecule described herein.

The antibiotic may vary widely, and is limited only by its ability to bechemically linked to the quorum-sensing molecule and by its ability toinactivate or reduce the pathogenicity of the targeted bacteria. In oneembodiment, the antibiotic is selected from the group consisting ofciprofloxacin, gentamicin, tobramycin, clarithromycin, piperacillin, andcombinations thereof. In another embodiment, the antibiotic isciprofloxacin. One non-limiting example of a quorum-sensingmolecule-antibiotic conjugate (also known as a “cip-conjugate”) isdepicted in (V):

wherein the —O represents a methoxy group. In the conjugate depicted in(V), the quorum-sensing molecule (II) has been conjugated to anantibiotic, ciprofloxacin. It is to be understood, however, that thestructures depicted above in (III) and (IV) may be conjugated to theantibiotic.

EXAMPLES

-   Synthesis of homoserine lactone (AHL-2), analogs (QS1207; QS0108);    (S)-Homoserine lactone hydrochloride: (S)-homoserine (25 g, 0.21    mol) was dissolved in aqueous hydrochloric acid (2.4 M, 322 mL, 0.97    mol, 4.6 equiv). The solution was refluxed for 3 h, and stirred at    ambient temperature overnight. Most of the solvent was removed    azeotropically with ethanol. Following crystal formation the    solution was cooled on ice. The resulting solid was filtered and    rinsed three times with cold ethanol. The filtrate was concentrated    and cooled, producing additional homoserine lactone. The process was    repeated 2 more times. After leaving the white powder on high vacuum    line overnight, 24 g (83% yield.) of homoserine lactone    hydrochloride was obtained. ¹H NMR (D2O) δ 4.59 (t, J=9.2 Hz, 1H),    4.43 (m, 2H), 2.76 (m, 1H), 2.42 (m, 1H), ¹³C NMR (D2O) δ 178.3,    68.3, 49.4, 27.6.-   (S)—N-butyryl homoserine lactone (AHL-2): (S)-homoserine lactone    (0.100 g, 0.726 mmol) was dissolved in 5 mL of acetonitrile.    N,N-diisopropylethyl amine (0.32 mL, 1.8 mmol) was added, followed    by butyrylchloride (0.15 mL, 1.4 mmol). The reaction was stirred    overnight. The acetonitrile was evaporated and the crude product was    purified by flash chromatography using 80% ethyl acetate-hexanes.    0.1 g of white crystals was obtained (81% yield) (FIG. 1; Scheme 1).    ¹H NMR (CDCl₃) δ 6.72 (d, J=6.2 Hz, 1H), 4.58 (m, 1H), 4.42 (app t,    J=8.1 Hz, 1H), 4.24 (m, 1H), 2.71 (m, 1H), 2.20 (m, 3H), 1.63 (sex,    J=7.4 Hz, 2H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (CDCl3) δ 175.8,    173.6, 65.9, 48.8, 37.8, 29.7, 18.8, 13.5 (16)-   (S)—N-butyryl homocysteine thiolactone (QS0108): (S)-homocysteine    thiolactone (0.23 g, 1.5 mmol) was added to 5 mL of methylene    chloride. N,N-diisopropylethyl amine (0.32 mL, 1.8 mmol) was added,    followed by butyrylchloride (0.15 mL, 1.4 mmol), at ambient    temperature. The reaction was stirred overnight. The reaction    mixture was then evaporated and the crude product was purified by    flash chromatography using 30% ethyl acetate hexanes. 0.14 g of pale    yellow white crystals were obtained (50% yield) (FIG. 1; Scheme 2),    ¹H NMR (CDCl3) δ 6.56 (d, J=6 Hz, 1H), 4.6 (m, 1H), 3.33 (m, 1H),    3.23 (m, 1H), 2.78 (m, 1H), 2.20 (t, J=7 Hz, 2H), 1.97 (m, 1H), 1.66    (sex, J=7 Hz, 2H), 0.93 (t, J=7 Hz, 3H). ¹³C NMR (CDCl3) δ 205.9,    173.7, 59.7, 38.5, 32.4, 27.8, 19.1, 13.8.-   (S)—N-Thiobutyryl homocysteine thiolactone (QS1207): The    (S)—N-butyryl homoserine lactone (0.97 g, 5.6 mmol) was placed in a    100 mL round bottom flask and 50 mL of toluene (stock) was added.    The flask was then fitted with a reflux condenser and purged with    argon. Lawesson's reagent (2.26 g, 5.6 mmol) was then added as a    solid. The suspension was stirred until homogenous and brought to    reflux with a heating mantle. The reaction was periodically    monitored by TLC (35% ethyl acetate/hexane; v/v). After 4 hours the    reaction was cooled and the volume was reduced to around 3 mL.    Purification by flash column chromatography gave rise to a    crystalline material (0.3 g, 26% yield) (FIG. 1; Scheme 3). ¹H NMR    (CDCl3) δ 7.60 (bs, 1H), 4.6 (m, 1H), 5.15 (m, 1H), 3.31 (m, 2H),    2.70 (1, J=7 Hz, 2H), 1.95 (m, 1H), 1.90 (m, 2H), 0.96 (t, J=7 Hz,    3H), ¹³C NMR (CDCl3 δ=77.0) δ 207.6, 205.2, 64.0, 48.2, 30.2, 27.5,    22.6, 13.1. (FIG. 1, Scheme 3).-   Synthesis of conjugate (QS0108-Cip):    (D,L)-4-bromo-N-(2-oxotetrahydrothiophen-3-yl) butanamide.    D,L-homocysteine thiolactone hydrochloride (5.2 g, 34 mmol) was    dissolved in water and covered with ethyl acetate. The solution was    cooled to 0° C. and the acid chloride (7.06, 38 mmol) was added    dropwise. Subsequently 1N NaOH was added dropwise (44.2 mmol). The    mixture was stirred for 1 h during which the temperature of the    reaction warmed to ambient temperature. No precipitate was observed,    however, TLC analysis (10% MeOH/methylene chloride; v/v) indicated    all the starting material (dissolved in saturated NaHCO₃ before    spotting the TLC) had been consumed. The reaction mixture was    extracted several times with ethyl acetate. The ethyl acetate was    dried over Na₂SO₄, filtered, and the solvent was removed in vacuo to    give 2.2.4 g of an off-white solid (25% yield).-   Ciprofloxacin conjugate to    (D,L)-4-bromo-N-(2-oxotetrahydrothiophen-3-yl)butanamide: In a 25 mL    round bottom flask, under argon, was placed the    (S)-4-bromo-N-(2oxotetrahydrothiophen-3-yl)butanamide and the methyl    ester of Cip. 10 mL of acetonitrile was added. The solution was    stirred for 5 min then the K₂CO₃ was added. The mixture was then    stirred for 24 h at ambient temperature. The solution was then    warmed and stirred for an additional 24 h. The volatile components    were removed and the materials subjected to purification by column    chromatography to give an off white powder. ¹H NMR (CDCl3) δ. HRMS    C26H32O5N4F1 S1 531.20667 (error +/−0.992 ppm). Ciprofloxacin was    obtained from Sigma Aldrich (St. Louis, Mo.). Synthesis of    N-(2-oxtotetrahydrofuran-3-yl)butaneselenoamide. In a single necked    14/20 round bottom flask was placed a magnetic stir bar, 0.5000 g of    N-(2-oxotetrahydrofuran-3-yl)butyramide (2.920 mmol), and 1.000 g of    Woolins reagent (1.88 mmol). Dry toluene (20 mL) was then added. The    flask was then fitted with a reflux condenser and placed under    argon. The system was purged with argon for ˜30 min. The mixture was    then brought to a gentle reflux and stirred overnight. The reaction    was cooled and the solution was then added to silica gel column for    purification. Using a 30% ethyl acetate/hexane mixture (v/v) 0.330 g    of the selenoamide was isolated. Yield=48.2%. ¹H d (CDCl₃) 0.97 (t,    J=7 Hz, 3H), 1.86 (s, J=7.5 Hz, 1H), 2.17 (m, 1H), 2.75 (dd, J=1.8,    6.3 Hz, 2H), 3.25 (m, 1H), 4.40 (m, 1H), 4.53 (t=9.3 Hz, 1H), 8.8    (bs, 1H). ¹³C d (CDCl₃) 215.0, 175.2, 66.9, 57.2, 52.4, 29.3, 23.3,    13.3. ⁷⁷Se d (CDCl₃) 304.4.-   Bacterial strains: Pseudomonas aeruginosa PAO-1 (wild type; #BAA47)    was obtained from ATCC (Manassas, Va.). Pseudomonas mutant strains    JP-1 (−/−C12HSL); PDO-100 (−/−C4HSL) and JP-2 (−/C₁₂HSL & C₄HSL)    were obtained from Dr. B. Iglewski, University of Rochester, N.Y.    Strains of P. aeruginosa and B. cepacia were grown at 37° C. with    shaking in LB medium. Pseudomonas broth (ref) was used for    production of pyocyanin. Wild type and mutant strains were stored at    −80° C.; in glycerol stock. Unless otherwise stated in all    experiments bacteria were grown to their log phase (O.D 0.4 at A600;    corresponding CFU 5×10⁷/mL) and then inoculated into the medium    containing either 10 μM of synthetic autoinducer homoserine lactone    (C₄HSL; AHL-2) or the analogs (QS1207 & QS0108).-   Chemicals for AHL synthesis: All chemicals were purchased from Sigma    Aldrich (St. Louis, Mo.)-   Biofilm formation assay: Static biofilms were formed on chambered    cover glass (#1 borosilicate) by allowing growth of the bacteria for    48 hrs at 37° C. Chambers were inoculated with overnight grown    culture containing about 5×10⁷ bacteria, and were then supplemented    with either 10 μM. AHL-2 or QS-1207 or QS-0108. Biofilms were    stained by a BacLight™ kit (Invitrogen) and were imaged by an    Olympus™ Confocal Microscope (FV300), Olympus™ CCD camera and images    were analyzed by Olympus Fluoview™ software.-   Motility assays: Wild type and mutant strains of bacteria were    allowed to grow for 24 hrs in presence of either AHL-2 or the    analogs QS-1207 or QS-0108 and then examined for changes in    twitching motility and flagellar motility. For twitching motility    studies, plates with LB agar (1%) were poured to an average depth of    3 mm and dried briefly. The strains to be tested were stab    inoculated to the bottom of the petri dish and incubated for 24 hrs    at 37° C. After the incubation period the zone between the agar and    the bottom of the petri dish (the “twitch zone”), was measured as    described in Glessner, A. et al., Journal of bacteriology, 181,    1623-1629 (1999). Flagellum mediated motility was assayed by    inoculating the treated bacteria to the center of a LB agar (0.3%)    plate. After 24 hrs of incubation the plates were inspected for    radial zones of bacterial growth (swim zone) indicating a motile    response.-   Protease activities: Total protease production of AHL-2 and analog    treated wild type and mutant strains were assayed on skim milk agar    plate containing blood agar base. Ultra high temperature treated    skim milk was added to sterilized blood agar base. Bacterial    cultures after overnight treatment with AHL-2 and QS analogs were    deposited on milk agar plate. Following incubation with bacteria,    the diameters of the clear zones were measured as an indicator of    proteolytic activities as described in Reimmann, C. et al. Molecular    microbiology, 24, 309-319 (1997).-   Pyocyanin assay: Cultures grown in pseudomonas broth as described in    Essar, D. W. et al, Journal of bacteriology 172, 884-900 (1990) to    maximize pyocyanin production, were extracted with 3 ml of    chloroform and then re-extracted into 1 ml of 0.2N HCl to give a    deep red solution. The absorbance was measured at 520 nm, also as    described in Essar et al.-   Expression of pyocyanin mRNA: P. aeruginosa wild type (PAO-1) and    mutant strains (JP-1. PDO-100 and JP-2) were grown in the presence    of either AHL-2, QS-1207 or QS-0108 and RNA sampling points were    established. Total RNA from three independent cultures were    extracted using QIAGEN™ kit. A preliminary phase of destruction of    bacterial envelope was achieved by incubating in a solution of    lysozyme (1 mg/ml) for 3-5 min at room temperature. One step    quantitative real time RT-PCR was used to quantitate mRNA. Primer    for Phz1 gene (a key player in pyocyanin biosynthesis) was obtained    from Sigma Aldrich (St. Louis, Mo.) following the template published    by Lenz, A. P. et al., Applied and environmental microbiology, 74,    4463-4471 (2008). Primer and probe concentrations were determined by    performing the optimization protocols recommended by the    manufacturer TaqMan™ SYBR Green based RT-PCR was used to measure the    house keeping 16sRNA gene. Each sample was assayed in triplicate and    statistical analysis was done by two-tailed Mann-Whitney test.-   Antimicrobial efficacy testing: The minimal inhibitory concentration    (MIC) of free ciprofloxacin (Cip) vs. QS-0108 conjugated    ciprofloxacin (QS-0108-Cip) in logarithmic planktonic growth of wild    type P. aeruginosa PAO-1 was tested according to standard NCCLS    microdilution method (22). Exponentially growing bacteria were    incubated with free vs. conjugated Cip at concentrations ranging    from 1-50 μM for 3 h.-   Assessment of biofilm disrupting activity of QS-0108-Cip conjugate:    The susceptibility of PAO-biofilms was tested by either pre or post    treatment of free Cip vs. QS-0108-Cip at sub MIC doses (10-25 μM) of    the conjugate. In the “pre-treatment” group biofilm was initiated in    presence of free or conjugated Cip, whereas in the “post-treatment”    group drugs were applied after 24 hours of biofilm formation.    Biofilms were stained with Bac-Light Live/Dead stain at 48 hrs and    observed using confocal microscopy. In another experiment PAC)-1    biofilms were grown up to 72 hrs and then exposed to free vs.    conjugated Cip at 25 μM for 24 hrs. In a separate experiment    collagen treated chamber cover slips were coated with free vs.    conjugated Cip. The coverslips were washed to remove excess    unadhered antibiotics and PAO-1 biofilms were grown for 24 hr,    stained and visualized under a Zeiss™ epifluorescent microscope.    Quantification of these experiments was done by measuring CFU count.-   Viability and biofilm formation in bacteria treated with the    selenium-containing bacterial quorum-sensing molecule depicted    in (IV) above:-   Strains of Yersinia pestis, Yersinia enterocolitica and Yersinis    pseudomonas were grown for 48 hrs in LB medium containing either 10M    of AHL-1, 100 μM selenium alone or with the compound depicted in    (IV). All three control samples formed a dense multilayer biofilms    of varying thickness. Cultures treated with free selenium had no    notable inhibitory effect on biofilm formation or growth of the    bacteria. In contrast, treatment of these bacteria with (IV)    significantly inhibited biofilm formation and viability of    biofilm-growing bacteria. The cultures treated with (IV) formed    relatively thin biofilms, if any at all, and lacked the    three-dimensional architecture compared to control untreated biofilm    cultures. These results indicate that the biofilm inhibition    observed in bacterial samples treated with (IV) was not due to    selenium but to the conjugate itself.-   Infection of human cells by Pseudomonas aeruginosa: Studies also    were performed with Pseudomonas aeruginosa in A549 human lung    epithelial cells. Cells were either treated with Pseudomonas    aeruginosa alone, pretreated with the selenium-containing compound    depicted in (IV), followed by exposure to Pseudomonas aeruginosa,    and treated with PA followed by post-treatment with (IV). An    increase in the viability of cells was observed in cells pre- and    post-treated with (IV). These results indicate that QS-0208    prevented the infection of cells by Pseudomonas aeruginosa and    appeared to protect the cells from Pseudomonas aeruginosa pre- or    post-exposure.

In all embodiments of the present invention, all ranges are inclusiveand combinable. All numerical amounts are understood to be modified bythe word “about” unless otherwise specifically indicated. All documentscited in the Detailed Description of the invention are, relevant part,incorporated herein by reference; the citation of any document is not tobe construed as an admission that it is prior art with respect to thepresent invention. To the extent that any meaning or definition of aterm in this document conflicts with any meaning or definition of thesame term in a document incorporated by reference, the meaning ordefinition assigned to that term in this document shall govern.

Whereas particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A method of reducing bacterial pathogenicity comprising: a) providinga biological system comprising pathogenic bacteria which produce anatural quorum-sensing molecule; b) providing a synthetic bacterialquorum-sensing molecule having the structure:

c) introducing the synthetic quorum-sensing molecule into the biologicalsystem comprising pathogenic bacteria.
 2. The method of claim 1, whereinthe pathogenic bacteria are selected from the group consisting ofPseudomonas aeruginosa, Yersinia pestis, Yersinia enterocolitca,Yersinia pseudotuberculosis, Burkholderia cepecia, and combinationsthereof.
 3. The method of claim 1, wherein the pathogenic bacteria isPseudomonas aeruginosa.
 4. A method of reducing bacterial pathogenicitycomprising: a) providing a biological system comprising pathogenicbacteria which produce a natural quorum-sensing molecule; b) providing asynthetic bacterial quorum-sensing molecule having the structure:

c) introducing the synthetic quorum-sensing molecule into the biologicalsystem comprising pathogenic bacteria.
 5. The method of claim 4, whereinthe pathogenic bacteria are selected from the group consisting ofPseudomonas aeruginosa, Yersinia pestis, Yersinia enterocolitca,Yersinia pseudotuberculosis, Burkholderia cepecia, and combinationsthereof.
 6. The method of claim 5, wherein the pathogenic bacteria isPseudomonas aeruginosa.